A. Tumor Growth and Invasion
Tumor growth and invasion into normal tissues is dependent upon an adequate blood supply (Folkman, Ann. N.Y. Acad. Sci. 401:212-227, 1982; Kerbel, Bioessays 13:31-36, 1991). Agents that target tumor blood supply have been shown to prevent or delay tumor formation, and to promote the regression or dormancy of established tumors in preclinical models. Thus, antibodies against the endothelial cell growth factor VEGF (vascular endothelial growth factor), which is produced at high levels by various types of tumors (Dvorak et al., J. Exp. Med. 174:1275-1278, 1991), antibodies to VEGF receptor 2, and soluble VEGF receptors all have been shown to reduce tumor growth in experimental animal models (Kendall and Thomas, Proc. Natl. Acad. Sci. U.S.A. 90:10705-10709, 1993; Kim et al., Nature 362:841-844, 1993; Skobe et al., Nat Med. 3:1222-1227, 1997). Antibodies to the integrin αvβ3, which is expressed at high levels by angiogenic blood vessels and permits endothelial cells to interact with components of the extracellular matrix, have been shown to disrupt ongoing angiogenesis on the chick chorioallantoid membrane and produce regression of human tumors transplanted into this site (Cheresh and Spiro, J. Biol. Chem. 262:17703-17711, 1987; Brooks et al., Cell 79:1157-1164, 1994).
A truncated form of tissue factor targeted to tumor vascular endothelium was demonstrated to initiate formation of intravascular clots and promote the regression of experimental tumors established in mice (Huang et al., Science 275:547-550, 1997). Angiostatin, a fragment of plasminogen (O'Reilly et al., Cell 79:315-328, 1994), and endostatin, a fragment of collagen XVIII (O'Reilly et al., Cell 88:277-285, 1997), are known to inhibit the proliferation of endothelial cells in vitro and to suppress neovascularization in vivo. Both compounds also inhibit the growth of a variety of tumors in mice, and upon repeated cycles of treatment, promote sustained tumor dormancy without inducing drug resistance (O'Reilly et al., Nat. Med. 2:689-692, 1996; Boehm et al., Nature 390:404-407, 1997).
Other multifunctional drugs that can also inhibit angiogenesis have displayed antitumor effects. These include Interleukin-12 (Voest et al., J. Natl. Cancer Inst. 87:581-586, 1995) the Interferon-γ Inducible Protein-10 (Angiolillo et al., J. Exp. Med. 182:155-162, 1995; Strieter et al., Biochem. Biophys. Res. Commun. 210:51-57, 1995; Sgadari et al., Proc. Natl. Acad. Sci. U.S.A. 93:13791-13796, 1996), the monokine induced by Interferon-γ (Sgadari et al., Blood 89:2635-2643, 1997), a fragment of prolactin (Clapp et al., Endocrinology 133:1292-1299, 1993), synthetic analogues of fumagillin (Ingber et al., Nature 348:555-557, 1990), thalidomide (D'Amato et al., Proc Natl Acad Sci U.S.A. 91:4082-4085, 1994), Platelet Factor-4 (Maione et al., Science 247:77-79, 1990), and thrombospondin (Good et al., Proc Natl Acad Sci U.S.A. 87:6624-6628, 1990; Weinstat-Saslow et al., Cancer Res. 54:6504-6511, 1994).
B. Calreticulin
Calreticulin was first identified in skeletal muscle sarcoplasmic reticulum. (Ostwald and MacLennan, J. Biol. Chem. 249 (3):974-979, 1974). Fifteen years later it was cloned and the N-terminus was sequenced. This led to the discovery that several groups had independently identified the molecule and had given it different names, including, “high-affinity Ca2+”, “calregulin”, “CRP55” and “calsequestrin-like protein” (Ostwald and MacLennan, J. Biol. Chem. 249 (3):974-979, 1974; Waisman et al., J. Biol. Chem. 260(3):1652-1660, 1985; Macer, D. R. J. & Koch, G. L. E. J. Cell. Sci. 91:61-70, 1988; Damiani et al., Biochem Biophys Res Commun 165(3):973-980, 1989; Treves et al., Biochem. J. 271:473-480, 1990). Each of these groups identified calreticulin through different means, but all identified its ability to bind Ca2+.
Although most studies have indicated that calreticulin resides predominantly within the lumen of the endoplasmic reticulum, calreticulin may also be found in other cellular compartments. For example, calreticulin was detected on the plasma membranes of lymphoblastoid cells (Newkirk and Tsoukas, J. Autoimmun. 5:511-525, 1992) and epidermal keratinocyte lines (Kawashima, et al., Dermatology 189 Suppl. 1:6-10, 1994). It was proposed to represent, or to be closely related in structure, to the C1q receptor found on endothelial cells, B cells, T cells and other cells (Chen et al., J. Immunol. 153:1430-1440, 1994). Calreticulin is also a constituent of lytic granules contained in cytotoxic T and NK cells from which it is released during cell lysis (Dupuis et al., J. Exp. Med. 177:1-7, 1993), and has been purified from the culture supernatant of several cell types (Booth and Koch, Cell 59:729-737, 1989; Eggleton et al., Clin. Immunol. Immunopathol. 72:405-409, 1994) and from normal human plasma (Sueyoshi et al., Thromb. Res. 63:569-575, 1991). Several observations support the notion that calreticulin can also be a target for autoimmune responses (Lux et al., J. Clin. Invest. 89:1945-1951, 1992; Meilof et al., J. Immunol. 151:5800-5809, 1993).
Since the initial identification and cloning, the structure of calreticulin has been characterized. Mammalian calreticulin is a 417 amino acid peptide from which the 17 N-terminal amino acids are cleaved upon translocation to the lumen of the endoplasmic reticulum (Smith and Koch, Embo. J 8(12):3581-3586, 1989). In addition to being found in the lumen of the endoplasmic reticulum, calreticulin has been found in the cytoplasm, in the nucleus of some cells, and in the extracellular matrix (Michalak et al., Biochem. J. 285:681-692, 1992). Further studies revealed that calreticulin has three distinct domains, the N-terminal domain, a middle domain and the C-terminal domain.
The mature calreticulin is composed of an N-terminal domain consisting of 180 amino acids that are highly conserved. Proposed three-dimensional models indicate that the domain contains eight anti-parallel β-strands. Furthermore, the N-terminal domain has been found to bind a number of molecules including the alpha subunit of integrin, Zn2+, and the DNA binding domain of steroid receptors (Nash et al., Mol. Cellular Biochem. 135:71-78, 1994).
The middle domain of calreticulin stretches from amino acid 180 to amino acid 280. It is proline rich and has also been termed the P-domain. This domain has been found to have a high affinity for Ca2+ and contains a nuclear localization signal (Baksh and Michalak, J. Biol. Chem. 266:21458-21465, 1991).
Following the P-domain is the C-domain. This last domain is highly acidic and contains an endoplasmic reticulum retention signal. The C-domain binds to Factor IX, Factor X, and prothrombin (See U.S. Pat. No. 5,426,097, to Stern et al.).
Calreticulin has also been found to be useful in wound healing (See U.S. Pat. No. 5,591,716, to Siebert et al.).